The growing emphasis on the state of the environment requires the development of accurate, inexpensive testing methods to detect organic contaminants in soils and water. The effective pollution management of our lakes, other waterways, subsoils and the subterranean water table demands regular and stringent testing. The effectiveness of such testing is enhanced when the tests are simple, inexpensive, can be operated on site and give a rapid result. In this way, the progress of the toxic clean-up from the waterway can be monitored to see if clean-up efforts are meeting existing state or federal guidelines. Very often soil assays must be made before land sales can be completed if the presence of a contaminant once existed on the site or suspected to be present. The detection of the adverse effects from leaking underground storage tanks can be enhanced if convenient tests can be routinely run on soil and water samples taken directly from near the buried storage tanks. The extent to which a toxic spill has penetrated and thus contaminated the subsoil and/or the groundwater can be more effectively determined, assayed and monitored if the subsoil and/or the groundwater can be collected, sampled and tested directly on-site.
Further, the safety of workers in enclosed working facilities which handle potentially harmful organic compounds could be enhanced if simple testing kits were available to test routinely and accurately for the presence of contaminants in the air or water being consumed by the work force. For example, factory air could easily be bubbled through an aqueous solution which in turn could be tested for the presence of the harmful organic compounds. Alternatively, absorptive charcoal badges as are known in the art and used in the workforce, could be worn by the workers, collected after a specified period, treated with suitable extraction liquid and assayed.
However, it has been difficult accurately to assess the presence, especially on a quantitative basis, of certain volatile non-polar organic compounds such as, for example, benzene, toluene, xylene, perchloroethylene and trichloroethylene, that have limited solubility in polar solvents e.g., in water. Some organic compounds are so volatile that even short delays in testing a sample can result in their evaporating from the sample completely, or at very least, sufficiently to significantly alter the true value of the actual contaminant concentration in a particular environment. This problem is exacerbated when the contaminant being tested for is non-polar and only minimally soluble in common polar solvents such as water.
Water is an important source for the purpose of detecting organic contaminants since large and small bodies of water, both above and below ground level are often found to contain various contaminants, and hence are the target of contaminant testing. In addition, water is an inexpensive, safely transportable, non-volatile solvent and can easily be used to wash contaminated soil samples, thus obtaining soluble and partially soluble organic contaminants in aqueous solution.
Quick, inexpensive and accurate qualitative and quantitative tests are needed for the detection of certain organic compounds directly from water or soil. The present invention is an improvement upon known environmental immunoassay tests involving the collection and sampling of water, soil, or air followed by the testing of the water, soil, or air samples, for various contaminants including volatile organic compounds. The improvement according to this invention comprises improving immunoassay accuracy by eliminating all separate sampling steps and testing samples, as collected, on site, for the presence of suspected contaminants. It is contemplated that the air may be tested by dissolving the air sample in a suitable solvent such as, e.g. water or methanol, or absorbing air in activated charcoal that is then extracted with suitable solvent such as, e.g. methanol.
Related immunoassays are disclosed in the commonly assigned and related U.S. patent application, Ser. No. 059,721 filed Jun. 9, 1987 and now abandoned, and its copending, commonly assigned continuation-in-part U.S. application Ser. No. 200,952 filed Jun. 1, 1988. As taught in these U.S. patent applications, many techniques are known for determining the presence of volatile organic compounds in the workplace and the environment, such as gas chromatography, mass spectrophotometry, and high performance liquid chromatography. One drawback to these detection and testing methods is the need to transport the collected samples to an off-site laboratory for further analysis. The vessels containing the samples to be tested must be carefully packaged and shipped to off-site laboratory facilities which in turn raises the overall testing costs. Even the least porous containers suffer the drawback of losing some of the contained volatile materials when the sample containers are opened and prepared for testing such as, e.g., during the preparation of dilutions, or transfer to testing receptacles. The volatile contaminants thus evaporate from the collected sample, inevitably adversely affecting the accuracy of the test result. Certain especially volatile contaminants may leave the sample altogether, resulting in false negative test results indicating a false absence of potentially hazardous compounds at a particular testing site, or drastically understating the true volatile contaminant concentration.
Immunoassays have been commonly used in connection with diagnostic testing, and in conjunction with monitoring drug levels in humans and various animals. However, the use of immunoassay techniques to ascertain the extent to which certain contaminants are present in the environment is relatively new. The use of immunoassays to detect aromatic ring-containing hydrocarbon compounds in soil and aqueous solutions has been disclosed in the aforementioned commonly assigned U.S. patent applications.
The immunoassays according to the invention of this application, are normally assembled in the form of field test kits to be brought directly to the site to be tested. Such tests and test kits are relatively inexpensive, and can be designed to give accurate and immediate results. Soil and aqueous samples suspected of containing various volatile organic contaminants, such as, for example, benzene, toluene, xylene, perchloroethylene and trichloroethylene may thus be tested immediately on-site, before any substantial evaporation of the hazardous volatile contaminant can occur. Therefore, immunoassay testing protocols according to this invention which allow for direct, inexpensive, on-site testing for volatile organic contaminants from samples as collected, which are suspected of containing such contaminants, provide superior overall volatile organic compound detection capabilities.
Further, highly volatile organic compounds, such as benzene, toluene, xylene, perchloroethylene and trichloroethylene begin evaporating to a substantial extent within the first few minutes following collection, from aqueous solutions in which they are only marginally soluble in the first instance. According to heretofore accepted testing methods commonly used in the art, sources suspected of containing the volatile organic compound being tested for are initially sampled, and are often further sampled into aliquots of various volumes in order to preserve the original sample in the event further testing is needed. This aliquot sampling allows the volatile organic compound being assayed for to evaporate from the sample. Further, it has heretofore been customary in the field of immunoassays that extremely small sample volumes when performing an immunologically based test yield the best results. These small volumes usually range between 10 and 500 microliters. In addition, separating such a small volume from the entirety of the sample and transferring this small amount to the ultimate immunoassay reaction vessel, results in another opportunity for volatile organic contaminants to escape from the sample being tested. As a result, a certain percentage of volatile compound evaporation during these aforementioned laboratory procedures is inevitable. Such evaporation, or volatile compound escape invariably affects the final accuracy of the test. Therefore, to conduct an accurate assay for volatile organic compounds, the assay must be initiated as soon as possible after collection.
The degree of volatilization of organic compounds during testing is affected by various ambient conditions during testing such as temperature, solubility of the volatile organic compound in the aqueous solution, and surface area of the sampling receptacle, or testing vessel, etc. While field testing must be conducted at ambient temperatures, the testing receptacles into which the sample is introduced have heretofore been conventionally chilled to temperatures between 2.degree. and 8.degree. C. in a specific attempt to reduce the evaporation of volatiles from the sample. As already discussed, the sample sizes have heretofore been usually restricted and limited to the relatively small volume capacity of the reaction receptacles designed to be used in the accepted testing procedures known in the art. The volume capacity of these receptacles is typically less than approximately 5 mls. It has heretofore been widely believed in the field of immunoassays that sample concentration, rather than sample size, determines sensitivity of the assay. In fact, most prior art immunoassay protocols call for a minimum total test volume in order to measure the analyte at its highest possible concentration when compared to the sample volume. The art has not heretofore appreciated the effect of the sample size, or volume, on the overall accuracy and sensitivity of the assay for volatile compounds. There has to now been concern in the field that the integrity of samples containing volatiles is compromised prior to analysis due to volatile evaporation from the sample solvent. Such volatile evaporation continues to remain a significant cause of inaccurate testing results.
As mentioned previously, the sample volume has oftentimes been limited by design. Conventional practice often calls for use of a sample volume which is equivalent to or less than the volume of the antibody-coated portion of the receptacle device. For example, the use of 4 to 5 ml. cuvettes are common in the immunoassay field. Usually, only the bottom of the cuvette, up to only the 1 ml. mark is coated with antibody. Under these conditions, it is common for samples of not more than about 0.5 to 1.0 ml. to be introduced into the cuvette and assayed. It has now been found according to this invention that, in principle, when assaying for volatile compounds, the volume of the test sample should not be constrained by the amount of antibody coating present at the bottom of the sample vessel nor should the size of the collection vessel deter direct testing in that vessel.
As this invention contemplates, one can coat only the lower portion of a relatively large volume sample collection receptacle with an immunologically coated surface while assaying a total volume of sample that exceeds, by at least about double, the volume that can be contained within the coated portion of the receptacle. Alternatively, if it is cost prohibitive to coat the receptacle with expensive antibody preparation due to the size of the receptacle, then antibody coated insertable insoluble solid-phase devices can be used. Such devices may be of any desired shape or size, but are commonly in the form of wands, sticks, finned sticks, paddles, balls, beads, hoops, loops, meshes, baskets, spiral, etc. The use of such inserts is also contemplated regardless of the area of the surface exposed in the sample collection receptacle. It is further contemplated that the antibody-coated insert devices have a sufficient density to sink to, and remain at the bottom of the collection vessel. When the coated insert device is in the form of a stick, paddle or wand, it is contemplated that the coated portion of the device is completely depressed in the collection vessel so that it comes into contact with, and is held against the bottom of the collection vessel. In this way, the sample volume assayed would cover the antibody coated portion of each insert device to a significant excess.